Pancreatic beta-cells secrete insulin in response to elevated blood glucose levels. Insulin amongst other hormones plays a key role in the regulation of the fuel metabolism. Insulin leads to the storage of glycogen and triglycerides and to the synthesis of proteins. The entry of glucose into muscles and adipose cells is stimulated by insulin. In patients who suffer from diabetes mellitus type I or LADA (latent autoimmue diabetes in adults, Pozzilli & Di Mario, 2001, Diabetes Care. 8:1460-1467) beta-cells are being destroyed due to autoimmune attack. The amount of insulin produced by the remaining pancreatic islet cells is too low, resulting in elevated blood glucose levels (hyperglycemia). In diabetes mellitus type II liver and muscle cells loose their ability to respond to normal blood insulin levels (insulin resistance). High blood glucose levels (and also high blood lipid levels) in turn lead to an impairment of beta-cell function and to an increase in beta-cell death. Interestingly, beta-cell regenerative processes like neogenesis and replication do not appear to compensate for the loss of beta cell mass in type II diabetics, thus causing a reduction in total beta-cell mass over time. Eventually the application of exogenous insulin becomes necessary in type II diabetics for an adequate control of blood glucose levels.
In type I diabetics, where beta-cells are being destroyed by autoimmune attack, treatments have been devised which modulate the immune system and may be able to stop or strongly reduce islet destruction (Raz et al., 2001, Lancet 358: 1749-1753; Chatenoud et al., 2003, Nat Rev Immunol. 3: 123-132; Homann et al., Immunity. 2002, 3:403-415). However, due to the relatively slow regeneration of human beta-cells such treatments can be more successful if they are combined with treatments that can stimulate beta-cell regeneration.
Diabetes is a very disabling disease, because today's common anti-diabetic drugs do not control blood sugar levels well enough to completely prevent the occurrence of high and low blood sugar levels. Frequently elevated blood sugar levels are toxic and cause long-term complications like for example nephropathy, retinopathy, neuropathy and peripheral vascular disease. Extensive loss of beta cells also leads to deregulation of glucagon secretion from pancreatic alpha cells which contributes to an increased risk of dangerous hypoglycemic episodes. There is also a host of related conditions, such as obesity, hypertension, heart disease and hyperlipidemia, for which persons with diabetes are substantially at risk.
Apart from the impaired quality of life for the patients, the treatment of diabetes and its long term complications presents an enormous financial burden to our healthcare systems with rising tendency. Thus, for the treatment of diabetes mellitus type I and LADA, but also for the treatment of late stages of diabetes mellitus type II there is a strong need in the art to identify factors that induce regeneration of pancreatic insulin producing beta-cells. These factors could restore normal function of the endocrine pancreas once its function is impaired or event could prevent the development or progression of diabetes type I, LADA or diabetes type II.
Neurturin is a secreted protein which is expressed in embryonic pancreas. Recombinant neurturin has been shown to stimulate the differentiation of mouse embryonic stemcells into insulin producing cells. Moreover, transgenic mice with elevated neurturin levels in the pancreas have a substantially increased pancreatic beta-cell mass. Based on these findings, the use of neurturin for the treatment of pancreatic disorders such as diabetes has been proposed (see for example WO 03/99318 and WO 2005/051415, the disclosure of which is herein incorporated by reference).
Neurturin is a member of the GDNF family of ligands (GFL) comprised of Glial cell line-derived neurotrophic factor (GDNF), Neurturin, Artemin and Persephin. Mature neurturin is a homodimer with a size of 23.6 kDa comprised of 102 amino acid monomers. Each monomer contains 3 intra-chain disulfide bonds forming a cystine knot. An additional disulfide bridge is connecting the monomers.
Neurturin had previously been proposed as a treatment for neurodegenerative diseases such as Parkinsons, Alzheimers and Huntington's disease, motor neuron disorders, spinal cord injuries or hearing disorders (WO 97/08196, WO 99/06064; Akerud et al. J Neurochem. 1999; 73(1):70-78; Koeberle & Ball Neuroscience. 2002; 110(3):555-567; Bilak et al. Mol Cell Neurosci. 1999; 13(5):326-336; Perez-Navarro et al. Neuroscience. 2000; 98(1):89-96; Rosenblad et al, Eur J Neurosci. 1999; 11(5):1554-1566, the disclosures of which are herein incorporated by reference).
It was found, however, that upon convection-enhanced delivery (CED) into rat brains, the distribution volume of neurturin and the related factor GDNF was limited (Hamilton et al., Exp Neurol. 2001; 168(1):155-161). Proteins must typically be administered by injection. After injection most proteins are cleared rapidly from the body, necessitating frequent injections. In our own studies, we found that the bioavailability after subcutaneous and intravenous injection of neurturin in mammals is low. Only about 10 percent of subcutaneously injected neurturin enter the circulation. Consequently, it is difficult to achieve therapeutically useful blood levels of the proteins in patients.
Thus, there is a strong need to develop a neurturin variant with enhanced availability in patients and therewith the development of methods to prolong the circulating half-lives of protein therapeutics in the body—the bioavailability—so that neurturin as active agent does not have to be injected frequently in order to satisfy the needs of patients for “user-friendly” protein therapeutics.
Thus, the underlying problem of the present invention was to provide new variants and formulations of neurturin that enhance its bioavailability.
The problem is solved by providing the embodiments characterized in the claims.